(Application to Plants)
The improvement of plants (introduction of desirable characters thereto) has been greatly dependent on classic breeding methods of crossing with wild species or mutants. Most of the varieties for use in the culture of ornamental plants and vegetables have been created through such efforts made by breeders. However, breeding often does not progress at all since a gene source does not exist. In addition, because breeding generally requires a long period of time even if a gene source exits, the improving of plants with genetic engineering techniques has been positively tried recently. For example, the creating a variety having resistance against viral diseases by conventional breeding methods is accompanied with many difficulties, e.g., an appropriate gene source cannot be found, or crossing with a wild species is difficult. In addition, with conventional breeding methods it is almost impossible to achieve drastic improvements, such as the creation of a dwarf plant or a dramatic increase in the number of flowers, and the like by regulating the subtle balance of plant hormones Recently, recombinant DNA techniques and plant tissue cultivation techniques have advanced and it has become possible to create virus-resistant plants and dwarf plants using such techniques. Major strategies so far tried are as described below.
(1) Creation of Virus-Resistant Plants
1) Introduction of a Viral Coat Protein Gene
In 1986, Powel Abel et al. (Science 232, 738) created a plant into which the coat protein gene of tobacco mosaic virus (TMV) had been introduced, and this plant was demonstrated to be TMV-resistant. Since then, a number of similar reports have been made throughout the world with various combinations of plants and viruses. However, a transgenic plant obtained by this method exhibits resistance against only one virus whose coat protein gene has been introduced (or extremely allied species thereof). In addition, the degree of resistance is greatly influenced by the inoculation concentration.
2) Use of a Satellite RNA
In some viruses, there is a low molecular weight RNA called satellite RNA. Satellite RNA depends on the parent virus for its replication and, in many cases, inhibits the growth of the parent virus to thereby remarkably reduce the symptoms induced by the virus.
By utilizing this property, it is possible to create a virus-resistant plant. To date, plants resistant to cucumber mosaic virus (Harrisson et al, Nature 328, 799) and tobacco ringspot virus (Gerlach et al., Nature 328, 802) have been created by introducing the cDNA of a satellite RNA into plants. In China, a plant integrating the cDNA of the cucumber mosaic virus satellite RNA has already been subjected to a field test to put it for practical use (Saito et al, Theor. Appl. Genet. 83, 679). However, this method is applicable to only those viruses having a satellite RNA.
3) Use of an Antisense RNA
A total or a partial cDNA of a virus is integrated into a plant so that it is transcribed and expressed in the antisense direction. When this plant is infected with the target virus, it is thought that the antisense RNA transcribed and the nucleic acid of the virus form a complex (a double-stranded RNA) to thereby inhibit the synthesis of viral proteins. As a result, the growth of the virus is inhibited. However, viral RNA is abudantly present in cells and has a complicated higher structure. Thus, the formation of such a complex is not considered easy and the effect of this method is not as great as expected (Cuozzo et al., Bio/Technology 6, 549). Even if resistance to viruses has been achieved, such resistance is expected only against the virus from which the cDNA was derived (or extremely allied species thereof), as observed in the method using a coat protein.
4) Use of a Ribozyme
A ribozyme is an RNA having an activity of self-catalyzed cleaving. It is possible to design a base sequence for a ribozyme so that it specifically cleaves viral RNA when the RNA is transcribed and expressed in plant cells. Similar to an antisense RNA, a ribozyme must form a complex with viral nucleic acid in order to produce its effect. Although there have not been many successful cases, for example, Edington et al. have reported that this method was effective when targeting at tobacco mosaic virus ("Viral Gene and Plant Pathogenesis").
5) Introduction of a Non-Structural Protein Gene Recently, there have been reports on several viruses that a transgenic plant incorporating the total cDNA of a viral replication enzyme gene or the cDNA having a mutation exhibits a high resistance against viruses (Golemboski et al., Proc. Natl. Acad. USA 87, 6311; Carr et al., Virology 199, 439). However, there have been reported instances where the resistance obtained by this method is easily overcome by a virus of a different strain from that of the targeted virus (Zairlin et al., Virology 201, 200).
(2) Change of Plant Morphology by Varying an Endogenous Cytokinin Concentration
If plant morphologenesis can be artificially controlled, it is possible to improve a plant into a desirable morphology for humans. This is especially important for flower business. Recently, research concerning morphologenesis in higher plants has been rapidly advancing with molecular biological techniques.
The bacteria Agrobacterium rhizogenes which infects plants and induces hair root carries a giant plasmid called Ri plasmid. A part of this plasmid is integrated into a plant genome. It is reported that, when the three genes of rol A, B and C in Ri plasmid have been integrated separately in tobacco, various morphological changes are observed (Schmulling et al., EMBO J. 7, 2621). In particular, rol C has been found to be a gene which increases the amount of cytokinins, a kind of plant hormone (Estruch et al., EMBO J. 10, 2889). Transgenic plants created with this gene exhibit changes such as the shortening of internodes, the lowering of plant heights, extrusion of styles, an increase in the number of flowers, expedited flowering time and the like. Some enterprises have already been developing a rose variety with an increased number of flowers, a dwarf variety of prairie gentian, etc. utilizing rol C.
Recently, it has been reported that SAHH binds to cytokinins (a kind of plant hormone) in plants (Mitsui et al., Plant Cell Physiol. 34, 1089). SAHH is an enzyme which catalyzes the following reaction. EQU S-Adenosylhomocysteine (SAH)+H.sub.2 O.fwdarw.Adenosine+Homocysteine
Methylation in cells progresses in the presence of S-adenosylmethionine (SAM) as the methyl group donor irrespective of microorganisms, animals and plants. SAM, after supplying the methyl group, becomes SAH. Therefore, SAHH which is an enzyme that hydroylzes SAH controls the concentrations of SAM and SAH in living organisms. SAHH is a key enzyme playing an important role in the methylation reaction. Since there have been found some other proteins which bind to cytokinins, it is not clear whether SAHH is a receptor for cytokinins with which cytokinins directly exert their physiological activity. However, it is presumed that SAHH is deeply involved in the exertion of the physiological activity of cytokinins and that an endogeneous cytokinin concentration regulates the methylation reaction in which SAHH is involved. This is still a matter of conjecture, since the above-mentioned report provides no data on the effect resulted from the binding of SAHH to cytokinins. On the other hand, the results of Examples of the present invention suggest that the effect of the binding of SAHH to cytokinins is that SAHH concentration is regulates the concentration of endogenous cytokinins, as opposed to what is conjectured in the above article.
(Application to Animals)
Described below are materials currently used as therapeutic agents for viral diseases in order to inhibit the infection with or growth of animal viruses.
1) Vaccines
Vaccines are mainly preventive means to inactivate invaded viruses by utilizing antibody production by an animal's immune system against viral antigens. Recently, various vaccines have been improved making free use of rapidly advanced genetic engineering and protein engineering ("Molecular Virology Promoting Life Sciences", Ishihama et al. (eds.), 1992, Kyoritu Shuppan Co., Ltd.). Among viruses, however, there are a number of them which skillfully escape from the immune system and against which no vaccine is effective.
2) Interferons
Inferferons are proteins induced by a viral infection. Interferons not only act on peripheral cells to make them resistant to viruses, but also have divergent physiological activities. It is considered that interferons are one of cytokines. Although they have achieved some results in a clinical application as therapeutics for hepatitis C in Japan, their use is limited because of their serious side effects.
3) Nucleic Acid Analogues
Viral DNA/RNA synthetases and reverse transcriptases are inhibited by nucleic acid analogues. Those nucleic acid analogues which are put into actual use as anti-AIDS agents include dideoxythymidine (ddC), azidothymidine (AZT) and dideoxyinosine (ddI). They can be expected to exert great effects as medicines, but their side effects are also great in view of their modes of action.
4) Others
Research and development of antiviral agents today is focused upon antisense medicines and inhibitors against transcriptional control factors of viruses. Out of the former, a therapeutic agent developed by ISIS in the United States for treating viral diseases of the eyes induced by cytomegalovirus and herpes virus has already been tested clinically. The characteristic of antisense medicines is that one target is specifically aimed at. Other subjects of research include protease inhibitors which inhibit viral proteases.
5) SAHH Inhibitors
All of the antiviral agents so far described are targeted at viruses per se. However, for the inhibition of a virus continuously growing in cells, a considerable amount of an antiviral agent is necessary and yet counter-measures should also be taken to cope with possible mutation of the virus to escape from the agent or possible inactivation of the agent by the virus. On the other hand, there is an idea to inhibit viral growth by inhibiting enzymes in those cells which have been infected with the virus. One embodiment of this idea is an SAHH inhibitor, which induces methylation inhibition in cells by inhibiting the host cells' SAHH. As a result, the cap structure is inhibited to thereby inhibit the translational function of a target virus. Sufficient resistance to a virus has been confirmed in in vitro experiments at a concentration of SAHH inhibitors at which no phytotoxicity is observed (Wolfe et al, J. Med. Chem. 34, 2521). It is reported that SAHH inhibitors are particularly effective against (-) RNA viruses and double-stranded viruses and that they also inhibit some of (+) RNA viruses and DNA viruses. Recently, there have been reported that SAHH inhibitors are also effective against retroviruses such as HIV.